Bacillus thuringiensis is a gram-positive bacterium that produces proteinaceous crystalline inclusions during sporulation. These B. thuringiensis crystal proteins are often highly toxic to specific insects. Insecticidal activities have been identified for crystal proteins from various B. thuringiensis strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitoes, flies) and Coleoptera (beetles).
Individual B. thuringiensis crystal proteins, also called delta-endotoxins or parasporal crystals or toxin proteins, can differ extensively in their structure and insecticidal activities. These insecticidal proteins are encoded by genes typically located on large plasmids, greater than 30 mega Daltons (MDa) in size, that are found in B. thuringiensis strains. A number of B. thuringiensis toxin genes have been cloned and the insecticidal crystal protein products characterized for their specific insecticidal properties. Reviews of B. thuringiensis toxin genes and crystal proteins are available (for example, Höfte et al., 1989; Schnepf et al., 1998).
The insecticidal properties of B. thuringiensis have been long recognized, and B. thuringiensis strains have been incorporated in commercial biological insecticide products for over forty years. Commercial B. thuringiensis insecticide formulations typically contain dried sporulated B. thuringiensis fermentation cultures whose crystal proteins are toxic to various insect species.
Traditional commercial B. thuringiensis bio-insecticide products are derived from “wild-type” B. thuringiensis strains, i.e., purified cultures of B. thuringiensis strains isolated from natural sources. Newer commercial B. thuringiensis bio-insecticide products are based on genetically altered B. thuringiensis strains, such as the transconjugant B. thuringiensis strains described in U.S. Pat. Nos. 5,080,897 and 4,935,353.
Various B. thuringiensis strains have been classified based on the reactions of the B. thuringiensis flagella with antibodies. A B. thuringiensis strain whose flagella react with a unique antibody is classified as a unique serovar, and over thirty different B. thuringiensis serovars or subspecies have been described (DeBarjac and Frachon, 1990).
Each B. thuringiensis subspecies often produces unique types of insecticidal crystal proteins. For example, B. thuringiensis subspecies kurstaki produces crystal proteins of approximately 130 kilo Daltons (kD) and 70 kD in size that are toxic to caterpillars, whereas B. thuringiensis subspecies tezebriottis produces a crystal protein of about 72 kD which is toxic to beetles.
A characteristic of crystal proteins is their ability to coalesce to form crystals inside the B. thuringiensis mother cell. Upon lysis of the mother cell the proteins are released as crystals into the external environment. In addition, B. thuringiensis also produces non-crystal proteins that, in contrast to crystal proteins, are secreted by B. thuringiensis cells as soluble proteins into the culture medium. Secreted non-crystal proteins of B. thuringiensis include phospholipases, proteases, and β-lactamase that have little, if any, insecticidal activity. However, three secreted non-crystal proteins of B. thuringiensis designated Vip1, Vip2 and Vip3 have been reported to be toxic to coleopteran or lepidopteran insects (Estruch et al., 1996; U.S. Pat. No. 5,866,326; WO94/21795; WO96/10083). A non-crystal protein of B. thuringiensis designated CryV is reported to be toxic to lepidopteran insects (Kostichka et al., 1996). A number of Bacillus thuringiensis isolates producing extracellular secreted insecticidal protein toxins have been previously identified (U.S. Pat. No. 5,840,868; U.S. Pat. No. 5,849,870; U.S. Pat. No. 5,866,326; U.S. Pat. No. 5,872,212; U.S. Pat. No. 5,877,012; U.S. Pat. No. 5,888,801; U.S. Pat. No. 6,204,435; U.S. Pat. No. 6,242,669; U.S. Pat. No. 6,279,369). Such strains have all been shown to produce one or more of these VIP or CryV toxin proteins or closely related homologs. Surprisingly, the inventors herein disclose a new class of extracellular secreted insecticidal protein toxins that do not exhibit homology to the known VIP or CryV classes of proteins.
Comparisons of amino acid sequences indicate that the Vip1, Vip2, Vip3, WAR, MIS and CryV protein classes are not related to the proteins of the present invention. Further comparison shows that none of the one hundred thirty-seven, more or less, known insect-toxic proteins of B. thuringiensis (Crickmore et al., 1998), are related to the proteins of the present invention. In fact, no significant homology was found between the sequences of the proteins of the present invention and any of the thousands of protein sequences contained in the National Center for Genome Resources (GenBank), Santa Fe, N. Mex. A BLAST search identified only two proteins in the GenBank database that suggested a possible homology to TIC901. The Bacillus sphaericus Mtx2 insecticidal protein exhibited only a 21% amino acid sequence identity over a contiguous 135 amino acid sequence length when aligned with TIC901. A putative amino acid sequence that may be expressed from a Fowlpox virus genome exhibited only a 27% amino acid sequence identity over a contiguous 147 amino acid sequence length when aligned with TIC901.